Arp2/3 complex is an evolutionarily conserved assembly of seven subunits with a key role in cellular motility. In response to various signals it mediates the assembly of branched actin filament networks during cellular movements. We propose a range of complementary methods to learn how nucleotides and activating peptides influence the conformation of Arp2/3 complex and how Arp2/3 complex forms actin filament branches. (1) The Pollard and Almo labs will use X-ray crystallography and radiation foot printing to provide high resolution structures of active conformations of Arp2/3 complex to document the conformational changes required to activate the complex for nucleating actin filaments and map interactions between the protein components of branches including the side of the mother filament. (2) The Rosen lab will use NMR to analyze activator peptide binding to Arp2/3 complex and conformational changes induced by activator peptides and nucleotides. (3) The Hanein and Volkmann labs will use electron cryomicroscopy and single-particle analysis to identify individual subunits in a range of conformationaI states of Arp2/3 complex with various bound nucleotides and activating peptides. They will also reconstruct Arp2/3 complex bound to the side of an actin filament and the structure of actin filament branches formed by Arp2/3 complex. (4) The Li lab will prepare novel forms of yeast Arp2/3 complex with isotopically labeled subunits for NMR and tagged subunits for labeling with gold for electron microscopy. The Li lab will use mutagenesis to generate Arp2/3 complex with faulty nucleotide binding or hydrolysis or other defects to trap conformational states useful for analysis of structures, biochemical pathways and physiological mechanisms.